Biomedical subjects

Anand Swaroop

Publications and source records attributed to Anand Swaroop.

At least 37 records · Page 2Linked to original sources

PubMed2005

Mechanisms of aging in senescence-accelerated mice.

BACKGROUND: Progressive neurological dysfunction is a key aspect of human aging. Because of underlying differences in the aging of mice and humans, useful mouse models have been difficult to obtain and study. We have used gene-expression analysis and polymorphism screening to study molecular senescence of the retina and hippocampus in two rare inbred mouse models of accelerated neurological senescence (SAMP8 and SAMP10) that closely mimic human neurological aging, and in a related normal strain (SAMR1) and an unrelated normal strain (C57BL/6J). RESULTS: The majority of age-related gene expression changes were strain-specific, with only a few common pathways found for normal and accelerated neurological aging. Polymorphism screening led to the identification of mutations that could have a direct impact on important disease processes, including a mutation in a fibroblast growth factor gene, Fgf1, and a mutation in and ectopic expression of the gene for the chemokine CCL19, which is involved in the inflammatory response. CONCLUSION: We show that combining the study of inbred mouse strains with interesting traits and gene-expression profiling can lead to the discovery of genes important for complex phenotypes. Furthermore, full-genome polymorphism detection, sequencing and gene-expression profiling of inbred mouse strains with interesting phenotypic differences may provide unique insights into the molecular genetics of late-manifesting complex diseases.

Aging↗

PubMed2005

Strong association of the Y402H variant in complement factor H at 1q32 with susceptibility to age-related macular degeneration.

Using a large sample of cases and controls from a single center, we show that a T-->C substitution in exon 9 (Y402H) of the complement factor H gene is strongly associated with susceptibility to age-related macular degeneration, the most common cause of blindness in the elderly. Frequency of the C allele was 0.61 in cases, versus 0.34 in age-matched controls (P<1x10(-24)). Genotype frequencies also differ markedly between cases and controls (chi2=112.68 [2 degrees of freedom]; P<1x10(-24)). A multiplicative model fits the data well, and we estimate the population frequency of the high-risk C allele to be 0.39 (95% confidence interval 0.36-0.42) and the genotype relative risk to be 2.44 (95% confidence interval 2.08-2.83) for TC heterozygotes and 5.93 (95% confidence interval 4.33-8.02) for CC homozygotes.

Aged↗

PubMed2005

Toll-like receptor 4 variant D299G is associated with susceptibility to age-related macular degeneration.

Age-related macular degeneration (AMD) is a genetically heterogeneous disease that leads to progressive and irreversible vision loss among the elderly. Inflammation, oxidative damage, cholesterol metabolism and/or impaired function of retinal pigment epithelium (RPE) have been implicated in AMD pathogenesis. We examined toll-like receptor 4 (TLR4) as a candidate gene for AMD susceptibility because: (i) the TLR4 gene is located on chromosome 9q32-33, a region exhibiting evidence of linkage to AMD in three independent reports; (ii) the TLR4-D299G variant is associated with reduced risk of atherosclerosis, a chronic inflammatory disease with subendothelial accumulation; (iii) the TLR4 is not only a key mediator of proinflammatory signaling pathways but also linked to regulation of cholesterol efflux and (iv) the TLR4 participates in phagocytosis of photoreceptor outer segments by the RPE. We examined D299G and T399I variants of TLR4 in a sample of 667 unrelated AMD patients and 439 unrelated controls, all of Caucasian ancestry. Multiple logistic regression demonstrated an increased risk of AMD in carriers of the G allele at TLR4 residue 299 (odds ratio=2.65, P=0.025), but lack of an independent effect by T399I variant. TLR4-D299G showed an additive effect on AMD risk (odds ratio=4.13, P=0.002) with allelic variants of apolipoprotein E (APOE) and ATP-binding cassette transporter-1 (ABCA1), two genes involved in cholesterol efflux. Interestingly, the effect of TLR4, APOE and ABCA1 variants on AMD susceptibility was opposite to that of association with atherosclerosis risk. Our data provide evidence of a link between multiple diverse mechanisms underlying AMD pathogenesis.

Aged↗

PubMed2005

Nephrocystin-5, a ciliary IQ domain protein, is mutated in Senior-Loken syndrome and interacts with RPGR and calmodulin.

Nephronophthisis (NPHP) is the most frequent genetic cause of chronic renal failure in children. Identification of four genes mutated in NPHP subtypes 1-4 (refs. 4-9) has linked the pathogenesis of NPHP to ciliary functions. Ten percent of affected individuals have retinitis pigmentosa, constituting the renal-retinal Senior-Loken syndrome (SLSN). Here we identify, by positional cloning, mutations in an evolutionarily conserved gene, IQCB1 (also called NPHP5), as the most frequent cause of SLSN. IQCB1 encodes an IQ-domain protein, nephrocystin-5. All individuals with IQCB1 mutations have retinitis pigmentosa. Hence, we examined the interaction of nephrocystin-5 with RPGR (retinitis pigmentosa GTPase regulator), which is expressed in photoreceptor cilia and associated with 10-20% of retinitis pigmentosa. We show that nephrocystin-5, RPGR and calmodulin can be coimmunoprecipitated from retinal extracts, and that these proteins localize to connecting cilia of photoreceptors and to primary cilia of renal epithelial cells. Our studies emphasize the central role of ciliary dysfunction in the pathogenesis of SLSN.

Amino Acid Sequence↗

PubMed2005

Photoreceptors of Nrl -/- mice coexpress functional S- and M-cone opsins having distinct inactivation mechanisms.

The retinas of mice null for the neural retina leucine zipper transcription factor (Nrl-/-) contain no rods but are populated instead with photoreceptors that on ultrastructural, histochemical, and molecular criteria appear cone like. To characterize these photoreceptors functionally, responses of single photoreceptors of Nrl-/- mice were recorded with suction pipettes at 35-37 degrees C and compared with the responses of rods of WT mice. Recordings were made either in the conventional manner, with the outer segment (OS) drawn into the pipette ("OS in"), or in a novel configuration with a portion of the inner segment drawn in ("OS out"). Nrl-/- photoreceptor responses recorded in the OS-out configuration were much faster than those of WT rods: for dim-flash responses tpeak = 91 ms vs. 215 ms; for saturating flashes, dominant recovery time constants, tau(D) = 110 ms vs. 240 ms, respectively. Nrl-/- photoreceptors in the OS-in configuration had reduced amplification, sensitivity, and slowed recovery kinetics, but the recording configuration had no effect on rod response properties, suggesting Nrl-/- outer segments to be more susceptible to damage. Functional coexpression of two cone pigments in a single mammalian photoreceptor was established for the first time; the responses of every Nrl-/- cell were driven by both the short-wave (S, lambda(max) approximately 360 nm) and the mid-wave (M, lambda(max) approximately 510 nm) mouse cone pigment; the apparent ratio of coexpressed M-pigment varied from 1:1 to 1:3,000 in a manner reflecting a dorso-ventral retinal position gradient. The role of the G-protein receptor kinase Grk1 in cone pigment inactivation was investigated in recordings from Nrl-/-/Grk1-/- photoreceptors. Dim-flash responses of cells driven by either the S- or the M-cone pigment were slowed 2.8-fold and 7.5-fold, respectively, in the absence of Grk1; the inactivation of the M-pigment response was much more seriously retarded. Thus, Grk1 is essential to normal inactivation of both S- and M-mouse cone opsins, but S-opsin has access to a relatively effective, Grk1-independent inactivation pathway.

Animals↗

PubMed2005

High throughput screening of co-expressed gene pairs with controlled false discovery rate (FDR) and minimum acceptable strength (MAS).

Many exploratory microarray data analysis tools such as gene clustering and relevance networks rely on detecting pairwise gene co-expression. Traditional screening of pairwise co-expression either controls biological significance or statistical significance, but not both. The former approach does not provide stochastic error control, and the later approach screens many co-expressions with excessively low correlation. We have designed and implemented a statistically sound two-stage co-expression detection algorithm that controls both statistical significance (false discovery rate, FDR) and biological significance (minimum acceptable strength, MAS) of the discovered co-expressions. Based on estimation of pairwise gene correlation, the algorithm provides an initial co-expression discovery that controls only FDR, which is then followed by a second stage co-expression discovery which controls both FDR and MAS. It also computes and thresholds the set of FDR p-values for each correlation that satisfied the MAS criterion. Using simulated data, we validated asymptotic null distributions of the Pearson and Kendall correlation coefficients and the two-stage error-control procedure; we also compared our two-stage test procedure with another two-stage test procedure using the receiver operating characteristic (ROC) curve. We then used yeast galactose metabolism data to illustrate the advantage of our method for clustering genes and constructing a relevance network. The method has been implemented in an R package "GeneNT" that is freely available from the Comprehensive R Archive Network (CRAN): www.cran.r-project.org/.

Algorithms↗

PubMed2005

Cone-like morphological, molecular, and electrophysiological features of the photoreceptors of the Nrl knockout mouse.

PURPOSE: To test the hypothesis that Nrl(-)(/)(-) photoreceptors are cones, by comparing them with WT rods and cones using morphological, molecular, histochemical, and electrophysiological criteria. METHODS: The photoreceptor layer of fixed retinal tissue of 4- to 6-week-old mice was examined in plastic sections by electron microscopy, and by confocal microscopy in frozen sections immunolabeled for the mouse UV-cone pigment and colabeled with PNA. Quantitative immunoblot analysis was used to determine the levels of expression of key cone-specific proteins. Single- and paired-flash methods were used to extract the spectral sensitivity, kinetics, and amplification of the a-wave of the ERG. RESULTS: Outer segments of Nrl(-/-) photoreceptors ( approximately 7 mum) are shorter than those of wild-type (WT) rods ( approximately 25 mum) and cones ( approximately 15 mum); but, like WT cones, they have 25 or more basal discs open to the extracellular space, extracellular matrix sheaths stained by PNA, chromatin "clumping" in their nuclei, and mitochondria two times shorter than rods. Nrl(-/-) photoreceptors express the mouse UV cone pigment, cone transducin, and cone arrestin in amounts expected, given the relative size and density of cones in the two retinas. The ERG a-wave was used to assay the properties of the photocurrent response. The sensitivity of the Nrl(-/-) a-wave is at its maximum at 360 nm, with a secondary mode at 510 nm having approximately one-tenth the maximum sensitivity. These wavelengths are the lambda(max) of the two mouse cone pigments. The time to peak of the dim-flash photocurrent response was approximately 50 ms, more than two times faster than that of rods. CONCLUSIONS: Many morphological, molecular, and electrophysiological features of the Nrl(-/-) photoreceptors are cone-like, and strongly distinguish these cells from rods. This retina provides a model for the investigation of cone function and cone-specific genetic disease.

Animals↗

PubMed2005

Isolation and characterization of 1,3-dicapryloyl-2-linoleoylglycerol: a novel triglyceride from berries of Hippophae rhamnoides.

1,3-Dicapryloyl-2-linoleoylglycerol (1), a novel triglyceride, was isolated from berries of Hippophae rhamnoides. The structure was elucidated on the basis of MS, 1D and 2D NMR experiments including HMQC and HMBC. The metal chelating, free radical scavenging, and lipid peroxidation inhibiting properties of the compound were also estimated with particular reference to radiation protection. In case of metal chelation and superoxide ion scavenging, 1 showed maximum inhibition at 50 microg/ml (11%) and 100 microg/ml (55%), respectively, whereas in lipid peroxidation, 1 showed maximum inhibition (57%) at 2 mg/ml as compared to quercetin as a control.

Antioxidants↗

PubMed2004

Recessive NRL mutations in patients with clumped pigmentary retinal degeneration and relative preservation of blue cone function.

Mice lacking the transcription factor Nrl have no rod photoreceptors and an increased number of short-wavelength-sensitive cones. Missense mutations in NRL are associated with autosomal dominant retinitis pigmentosa; however, the phenotype associated with the loss of NRL function in humans has not been reported. We identified two siblings who carried two allelic mutations: a predicted null allele (L75fs) and a missense mutation (L160P) altering a highly conserved residue in the domain involved in DNA-binding-site recognition. In vitro luciferase reporter assays demonstrated that the NRL-L160P mutant had severely reduced transcriptional activity compared with the WT NRL protein, consistent with a severe loss of function. The affected patients had night blindness since early childhood, consistent with a severe reduction in rod function. Color vision was normal, suggesting the presence of all cone color types; nevertheless, a comparison of central visual fields evaluated with white-on-white and blue-on-yellow light stimuli was consistent with a relatively enhanced function of short-wavelength-sensitive cones in the macula. The fundi had signs of retinal degeneration (such as vascular attenuation) and clusters of large, clumped, pigment deposits in the peripheral fundus at the level of the retinal pigment epithelium (clumped pigmentary retinal degeneration). Our report presents an unusual clinical phenotype in humans with loss-of-function mutations in NRL.

Amino Acid Motifs↗

PubMed2004

Recruitment of the rod pathway by cones in the absence of rods.

In mammalian retinas, rods and cones connect to distinct sets of bipolar cells. In the retina of Nrl (neural retina leucine zipper) knock-out mice, in which rods fail to form and all photoreceptors are cones, rod bipolar cells have normal morphology, pattern of staining, and lamination, but they form synaptic connections with cones. Hence, retinal interneurons are not entirely committed to the choice of their synaptic partners; also, their morphology and pattern of distribution of their processes in the synaptic layers of the retina are not instructed by the establishment of synaptic connections with cognate photoreceptor(s). These findings are of considerable relevance for our understanding of mechanisms responsible for cell recognition during development and in the context of using transplanted tissue to restore vision in retinal degeneration.

Animals↗

PubMed2004

The minimal transactivation domain of the basic motif-leucine zipper transcription factor NRL interacts with TATA-binding protein.

The basic motif-leucine zipper (bZIP) transcription factor NRL controls the expression of rhodopsin and other phototransduction genes and is a key mediator of photoreceptor differentiation. To delineate the molecular mechanisms underlying transcriptional initiation of rod-specific genes, we characterized different regions of the NRL protein using yeast-based autoactivation assays. We identified 35 amino acid residues in the proline- and serine-rich N-terminal region (called minimal transactivation domain, MTD), which, when combined with LexA or Gal4 DNA binding domains, exhibited activation of target promoters. Because this domain is conserved in all proteins of the large Maf family, we hypothesized that NRL-MTD played an important role in assembling the transcription initiation complex. Our studies showed that the NRL protein, including the MTD, interacted with full-length or the C-terminal domain of TATA-binding protein (TBP) in vitro. NRL and TBP could be co-immunoprecipitated from bovine retinal nuclear extract. TBP was also part of c-Maf and MafA (two other large Maf proteins)-containing complex(es) in vivo. Our data suggest that the function of NRL-MTD is to activate transcription by recruiting or stabilizing TBP (and consequently other components of the general transcription complex) at the promoter of target genes, and a similar function may be attributed to other bZIP proteins of the large Maf family.

Amino Acid Motifs↗

PubMed2004

Altered expression of genes of the Bmp/Smad and Wnt/calcium signaling pathways in the cone-only Nrl-/- mouse retina, revealed by gene profiling using custom cDNA microarrays.

Many mammalian retinas are rod-dominant, and hence our knowledge of cone photoreceptor biology is relatively limited. To gain insights into the molecular differences between rods and cones, we compared the gene expression profile of the rod-dominated retina of wild type mouse with that of the cone-only retina of Nrl(-/-) (Neural retina leucine zipper knockout) mouse. Our analysis, using custom microarrays of eye-expressed genes, provided equivalent data using either direct or reference-based experimental designs, confirmed differential expression of rod- and cone-specific genes in the Nrl(-/-) retina and identified novel genes that could serve as candidates for retinopathies or for functional studies. In addition, we detected altered expression of several genes that encode cell signaling or structural proteins. Prompted by these findings, additional real-time PCR analysis revealed that genes belonging to the Bmp/Smad and Wnt/Ca(2+) signaling pathways are expressed in the mature wild type retina and that their expression is significantly altered in the Nrl(-/-) retina. Chromatin immunoprecipitation analysis of adult retina identified Bmp4 and Smad4, which are down-regulated in the Nrl(-/-) retina, as possible direct transcriptional targets of Nrl. Consistent with these studies, Bmp4 and Smad4 are expressed in the mature rod photoreceptors of mouse retina. Modulation of Bmp4 and/or Smad4 by Nrl may provide a mechanism for integrating diverse cell signaling networks in rods. We hypothesize that Bmp/Smad and Wnt/Ca(2+) pathways participate in cell-cell communication in the mature retina, and expression changes observed in the Nrl(-/-) retina reflect their biased utilization in rod versus cone homeostasis.

Animals↗

PubMed2004

Nuclear receptor NR2E3 gene mutations distort human retinal laminar architecture and cause an unusual degeneration.

Mutations in the nuclear receptor gene, NR2E3, cause a disorder of human retinal photoreceptor development characterized by hyperfunction and excess of the minority S (short wavelength or blue) cone photoreceptor type, but near absence of function of the majority rod receptor. NR2E3 disease can also progress to blindness. How the human retina accommodates mis-specified types and numbers of neurons and advances to retinal degeneration are unknown. We studied the retinal organization in vivo of patients with NR2E3 mutations. Early human NR2E3 disease with S cone hyperfunction showed thickened retinal layers within an otherwise normally structured retina. With visual loss, however, lamination was coarse and there was a strikingly thick and bulging appearance to the retina, localized to an annulus encircling the central fovea. This pattern was not found in other retinal degenerations. The abnormal laminar retinal architecture of early NR2E3 disease may be due in part to larger cells with an S cone phenotype in place of rods that failed to differentiate. The later-stage dysplastic appearance suggests a previously unrecognized proliferative response in human retinal degeneration.

Humans↗

PubMed2004

Photoreceptor-specific nuclear receptor NR2E3 functions as a transcriptional activator in rod photoreceptors.

NR2E3, a photoreceptor-specific orphan nuclear receptor, is believed to play a pivotal role in the differentiation of photoreceptors. Mutations in the human NR2E3 gene and its mouse ortholog are associated with enhanced S-cones and retinal degeneration. In order to gain insights into the NR2E3 function, we performed temporal and spatial expression analysis, yeast two-hybrid screening, promoter activity assays and co-immunoprecipitation studies. The Nr2e3 expression was localized preferentially to the rod, and not to the cone, photoreceptor nuclei in rodent retina. The yeast two-hybrid screening of a retinal cDNA library, using NR2E3 as the bait, identified another orphan nuclear receptor NR1D1 (Rev-erbalpha). The interaction of NR2E3 with NR1D1 was confirmed by glutathione S-transferase pulldown and co-immunoprecipitation experiments. In transient transfection studies using HEK 293 cells, both NR2E3 and NR1D1 activated the promoters of rod phototransduction genes synergistically with neural retina leucine zipper (NRL) and cone-rod homeobox (CRX). All four proteins, NR2E3, NR1D1, NRL and CRX, could be co-immunoprecipitated from the bovine retinal nuclear extract, suggesting their existence in a multi-protein transcriptional regulatory complex in vivo. Our results demonstrate that NR2E3 is involved in regulating the expression of rod photoreceptor-specific genes and support its proposed role in transcriptional regulatory network(s) during rod differentiation.

Animals↗

PubMed2004

Expression profiling of the developing and mature Nrl-/- mouse retina: identification of retinal disease candidates and transcriptional regulatory targets of Nrl.

The rod photoreceptor-specific neural retina leucine zipper protein Nrl is essential for rod differentiation and plays a critical role in regulating gene expression. In the mouse retina, rods account for 97% of the photoreceptors; however, in the absence of Nrl (Nrl-/-), no rods are present and a concomitant increase in cones is observed. A functional all-cone mouse retina represents a unique opportunity to investigate, at the molecular level, differences between the two photoreceptor subtypes. Using mouse GeneChips (Affymetrix), we have generated expression profiles of the wild-type and Nrl-/- retina at three time-points representing distinct stages of photoreceptor differentiation. Comparative data analysis revealed 161 differentially expressed genes; of which, 78 exhibited significantly lower and 83 higher expression in the Nrl-/- retina. Hierarchical clustering was utilized to predict the function of these genes in a temporal context. The differentially expressed genes primarily encode proteins associated with signal transduction, transcriptional regulation, intracellular transport and other processes, which likely correspond to differences between rods and cones and/or retinal remodeling in the absence of rods. A significant number of these genes may serve as candidates for diseases involving rod or cone dysfunction. Chromatin immunoprecipitation assay showed that in addition to the rod phototransduction genes, Nrl might modulate the promoters of many functionally diverse genes in vivo. Our studies provide molecular insights into differences between rod and cone function, yield interesting candidates for retinal diseases and assist in identifying transcriptional regulatory targets of Nrl.

Animals↗

PubMed2004

Connexin 36 in photoreceptor cells: studies on transgenic rod-less and cone-less mouse retinas.

PURPOSE: Rod-cone gap junctions permit transmittal of rod visual information to the cone pathway. A recent report has shown that this transfer does not occur in mice in which the gap junction protein connexin 36 is knocked out indicating that rod-cone gap junctions are assembled from this protein. It remains unresolved, however, whether rods, cones or both express connexin 36. We have tried to address this question with the use of transgenic rod-less and cone-less mice. METHODS: Deletion of Nrl, a transcription factor, results in a complete loss of rods with a concomitant increase in S-cones. We used this as the rod-less (cone-only) model. Cone-less (rod-only) retinas were from mice expressing an attenuated diphtheria toxin gene under the control of a promoter selective for cones. Nearly all long wavelength cones and 95% of short wavelength cones are missing in this model. Fixed retinal sections from these two models and age matched controls were used to detect connexin 36 gap junctions by immunofluorescence. RESULTS: Punctate immunofluorescence, indicating the presence of gap junctions, was observed in the inner and outer plexiform layers of both wild type and cone-less and rod-less retinas. Our assumption was that immunofluorescence due to photoreceptor gap junctions would be observed in the outer plexiform layer. In all the animals, most of the immunofluorescence was in the inner plexiform layer, with only a marginal reaction in the outer plexiform layer. In cone-only (rod-less) retina, immunofluorescence in the outer plexiform layer increased by more than 20 fold compared to wild type. In rod-only (cone-less) retina, the outer plexiform layer showed about a 30% decrease in immunofluorescence. In both rod-less and cone-less retinas, immunofluorescence in the inner plexiform layer was higher than in the wild type by 25-50%. CONCLUSIONS: Cones constitute only about 3% of photoreceptors in the wild type retina while they make up 100% of the photoreceptors in cone-only retina. This increase in their numbers coincided with a 20 fold increase in immunofluorescence in the outer plexiform layer, strongly suggesting that cones express connexin 36. Conversely, when the cone numbers went down from 3% to near zero in cone-less retina, immunofluorescence decreased by about 30% in the outer plexiform layer, suggesting again that cones express the connexin and that they contribute to its presence disproportionately more than their numbers indicate. The results from both rod-less and cone-less animals are strongly indicative of cones expressing connexin 36, but are not sufficient to conclude whether rods express the protein. An unexpected observation from our experiments is that immunofluorescence increases slightly in the inner plexiform layer in both rod-less and cone-less retina for reasons that need further investigation.

Animals↗

PubMed2004

QRX, a novel homeobox gene, modulates photoreceptor gene expression.

A novel paired-like homeobox gene, designated as Qrx, was identified by a yeast one-hybrid screen using the bovine Rhodopsin promoter Ret-1 DNA regulatory element as bait. Qrx is preferentially expressed in both the outer and inner nuclear layers of the retina. Its homeodomain is nearly identical to that of Rx/Rax, a transcription factor that is essential for eye development, but it shares only limited homology elsewhere. Although Qrx and Rx/Rax show similar DNA binding properties in vitro, the two proteins demonstrate distinct target selectivity and functional behavior in promoter activity assays. QRX synergistically increases the transactivating function of the photoreceptor transcription factors Crx and NRL and it physically interacts with CRX. Qrx is present in the bovine and human genomes, but appears to be absent from the mouse genome. Nonetheless, a 5.8 kb upstream region of human QRX is capable of directing expression in presumptive photoreceptor precursor cells in transgenic mice. These results indicate that Qrx may be involved in modulating photoreceptor gene expression. In addition, the finding of rare heterozygous QRX sequence changes in three individuals with retinal degeneration raises the possibility that QRX may be involved in disease pathogenesis.

Amino Acid Sequence↗

PubMed2004

Functional analysis of the rod photoreceptor cGMP phosphodiesterase alpha-subunit gene promoter: Nrl and Crx are required for full transcriptional activity.

To understand the factors controlling expression of the cGMP phosphodiesterase type 6 (PDE6) genes, we have characterized the promoter of the human PDE6A gene that encodes the catalytic alpha-subunit. In vivo DNase I hypersensitivity assays revealed two sites immediately upstream of the PDE6A core promoter region. Transient transfection assay in Y79 cells of constructs containing varying lengths of the promoter region showed a decrease in promoter activity with increasing length. The most active segment contained a 177-bp upstream sequence including apparent Crx and Nrl transcription factor binding sites. Both Crx and Nrl transactivated the PDE6A promoter in HEK293 cells and showed a >100-fold increase when coexpressed. Coexpression of a dominant negative inhibitor of Nrl abolished Nrl transactivation but had no effect on Crx. DNase I footprinting assays identified three potential Crx binding sites within a 55-bp segment beginning 29 bp upstream of the transcription start point. Mutation of two of these sites reduced reporter gene activity by as much as 69%. Gel shifts showed that all three Crx sites required a TAAT sequence for efficient binding. Consistent with a requirement for Crx and Nrl in Pde6a promoter activity, Pde6a mRNA is reduced by 87% in the retina of Crx(-/-) mice and is undetectable in Nrl(-/-) mice at postnatal day 10. These results establish that both Nrl and Crx are required for full transcriptional activity of the PDE6A gene.

3',5'-Cyclic-GMP Phosphodiesterases↗